Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E

Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment. © 2005 SGM.postprin

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

BACKGROUND: The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. METHODS: Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. RESULTS: A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. CONCLUSIONS: The novel coronavirus might have a role

Toll-like receptor 2 promiscuity is responsible for the immunostimulatory activity of nucleic acid nanocarriers.

Lipopolyamines (LPAs) are cationic lipids; they interact spontaneously with nucleic acids to form lipoplexes used for gene delivery. The main hurdle to using lipoplexes in gene therapy lies in their immunostimulatory properties, so far attributed to the nucleic acid cargo, while cationic lipids were considered as inert to the immune system. Here we demonstrate for the first time that di-C18 LPAs trigger pro-inflammatory responses through Toll-like receptor 2 (TLR2) activation, and this whether they are bound to nucleic acids or not. Molecular docking experiments suggest potential TLR2 binding modes reminiscent of bacterial lipopeptide sensing. The di-C18 LPAs share the ability of burying their lipid chains in the hydrophobic cavity of TLR2 and, in some cases, TLR1, at the vicinity of the dimerization interface; the cationic headgroups form multiple hydrogen bonds, thus crosslinking TLRs into functional complexes. Unravelling the molecular basis of TLR1 and TLR6-driven heterodimerization upon LPA binding underlines the highly collaborative and promiscuous ligand binding mechanism. The prevalence of non-specific main chain-mediated interactions demonstrates that potentially any saturated LPA currently used or proposed as transfection agent is likely to activate TLR2 during transfection. Hence our study emphasizes the urgent need to test the inflammatory properties of transfection agents and proposes the use of docking analysis as a preliminary screening tool for the synthesis of new non-immunostimulatory nanocarriers.MP would like to thank the FRIA-FNRS (F3/5/5-MCF/XH/FC-17514) for its financial support. NG and MG would like to thank the Wellcome Trust (WT100321/z/12/Z) for financial support. CL is grateful to the foundation Wiener Anspach and the Marie Curie Actions (TLR4-CAT PIEF-GA-2012-326481) for financial support

Cytosolic phospholipase A2α gene silencing in monocytes alters development of Th1 responses and reduces autoimmune arthritis

International audienc

Codon conservation in the influenza A virus genome defines RNA packaging signals

Genome segmentation facilitates reassortment and rapid evolution of influenza A virus. However, segmentation complicates particle assembly as virions must contain all eight vRNA species to be infectious. Specific packaging signals exist that extend into the coding regions of most if not all segments, but these RNA motifs are poorly defined. We measured codon variability in a large dataset of sequences to identify areas of low nucleotide sequence variation independent of amino acid conservation in each segment. Most clusters of codons showing very little synonymous variation were located at segment termini, consistent with previous experimental data mapping packaging signals. Certain internal regions of conservation, most notably in the PA gene, may however signify previously unidentified functions in the virus genome. To experimentally test the bioinformatics analysis, we introduced synonymous mutations into conserved codons within known packaging signals and measured incorporation of the mutant segment into virus particles. Surprisingly, in most cases, single nucleotide changes dramatically reduced segment packaging. Thus our analysis identifies cis-acting sequences in the influenza virus genome at the nucleotide level. Furthermore, we propose that strain-specific differences exist in certain packaging signals, most notably the haemagglutinin gene; this finding has major implications for the evolution of pandemic viruses

EVALUATION DE L'INTERET THERAPEUTIQUE DE LA GREFFE DE MOELLE OSSEUSE DANS LE CADRE DE LA MYOPATHIE DE DUCHENNE, ETUDE PRECLINIQUE DANS UN ELEVAGE CANIN

La myopathie de Duchenne est une affection musculaire létale de l'enfant causée par le déficit d'une protéine, la dystrophine. Malgré l'identification du gène en cause, cette maladie ne dispose d'aucun traitement curatif. Parmi les modèles animaux spontanés, si le chien GRMD et la souris mdx présentent une mutation sur le gène de la dystrophine et des caractéristiques biochimiques de dystrophinopathie, seul le chien, comme l'homme, exprime un phénotype grave. Ainsi, le chien dystrophique est considéré comme la meilleure " phénocopie " de la maladie humaine et se place comme un modèle " préclinique " incontournable dans l'évaluation de stratégies thérapeutiques. Le but de ce travail fut de tester, chez le chien dystrophique, l'intérêt clinique d'une voie thérapeutique prometteuse élaborées dans le modèle marin ; la thérapie cellulaire médiée par la greffe de moelle osseuse. Chez la souris mdx, les expériences de greffe de moelle osseuse ont montré que les cellules souches médullaires marines ont la capacité de renouveler très partiellement le tissu musculaire et donc de restaurer l'expression de la dystrophine. Nous avons retrouvé les mêmes potentialités myogéniques des cellules souches lors de greffe de moelle osseuse chez le chien dystrophique mais n'avons pu mettre en évidence de bénéfice clinique. Cette étude confirme le caractère prometteur de la greffe de cellules souches dans le cadre des dystrophies musculaires mais le faible nombre de fibres dystrophine positives observées au sein du muscle interdit toute application thérapeutique immédiate. Il est donc nécessaire de trouver un moyen de mobiliser ces cellules et de les attirer au sein du muscle pour atteindre un seuil thérapeutique. Cette études souligne également la nécessité d'améliorer les connaissances sur la physiopathogénie du muscle dystrophique en particulier les mécanismes qui inhibent la régénération musculaire.MAISONS-ALFORT-Ecole Vétérin (940462302) / SudocSudocFranceF